Differential gene expression in dairy cows under negative energy balance and ketosis: A systematic review and meta-analysis.

Development of ketosis in high-producing dairy cows contributes to several animal health issues and highlights the need for a better understanding of the genetic basis of metabolic diseases. To evaluate the pattern of differential gene expression in the liver of cows under negative energy balance (NEB), and under subclinical and clinical ketosis, a meta-analysis of gene expression and genome-wide association studies results was performed. An initial systematic review identified 118 articles based on the key words "cow," "liver," "negative energy balance," "ketosis," "expression," "qPCR," "microarray," "proteomic," "RNA-Seq," and "GWAS." After further screening for only peer-reviewed and pertinent articles for gene expression during NEB and clinical and subclinical ketosis (considering plasma levels of ß-hydroxybutyrate), 20 articles were included in the analysis. From the systematic review, 430 significant SNPs identified by genome-wide association studies (GWAS) were assigned to genes reported in gene expression studies by considering chromosome and base pair positions in the ARS-UCD 1.2 bovine assembly. Venn diagrams were created to integrate the data obtained in the systematic review, and Gene Ontology enrichment analysis was carried out using official gene names. A QTL enrichment analysis was also performed to identify potential positional candidate loci. Twenty-four significant SNPs were located within the coordinates of differentially expressed genes located on chromosomes 2, 3, 6, 9, 11, 14, 27, and 29. Three significant metabolic pathways were associated with NEB and subclinical and clinical ketosis. In addition, 2 important genes, PPARA (peroxisome proliferator activated receptor alpha) and ACACA (acetyl-coenzyme A carboxylase α), were identified, which were differentially expressed in the 3 metabolic conditions. The PPARA gene is involved in the regulation of lipid metabolism and fatty liver disease and the ACACA gene encodes an enzyme that catalyzes the carboxylation of acetyl-coenzyme A to malonyl-coenzyme A, which is a rate-limiting step in fatty acid synthesis. Gene network analysis revealed co-expression interactions among 34 genes associated with functions involving fatty acid transport and fatty acid metabolism. For the annotated QTL, 9 QTL were identified for ketosis. The genes FN1 (fibronectin 1) and PTK2 (protein tyrosine kinase 2), which are mainly involved in cell adhesion and formation of extracellular matrix constituents, were enriched for QTL previously associated with the trait "ketosis" on chromosome 2 and for the trait "milk iron content" on chromosome 14, respectively. This integration of gene expression and GWAS data provides an additional understanding of the genetic background of NEB and subclinical and clinical ketosis in dairy cattle. Thus, it is a useful approach to identify biological mechanisms underlying these metabolic conditions in dairy cattle.

Developmental ecdysteroid titers and DNA puffs in larvae of two sciarid species, Rhynchosciara americana and Rhynchosciara milleri (Diptera: Sciaridae).

Ecdysteroid titers, developmental landmarks and the presence of prominent amplifying regions (DNA puffs) have been compared during late larval to pupal development in four groups of Rhynchosciara americana larvae and in R. americana and Rhynchosciara milleri. Three prominent DNA puffs (B2, C3 and C8) expand and regress sequentially on the rising phase of the 20-hydroxyecdysone (20E) titer in R. americana as a firm, cellular cocoon is being constructed. A sharp rise in 20E coincides with the regression of these puffs. The shape of the 20E curve is similar in R. milleri, a species that does not construct a massive cocoon, but the behavior of certain DNA puffs and their temporal relationship to the curve differs. Regions corresponding to B2 and C3 can be identified in R. milleri by banding pattern similarity with R. americana chromosomes and, in the case of B2, by hybridization to an R. americana probe. A B2 puff appears in R. milleri as the 20E titer rises but remains small in all gland regions. A puff similar to the R. americana C3 puff occurs in posterior gland cells of R. milleri (C3(Rm)) after the B2 puff, but this site did not hybridize to R. americana C3 probes. C3(Rm) incorporated (3)H-thymidine above background, but showed less post-puff DNA accumulation than C3 of R. americana. R. americana C8 probes hybridized to a more distal region of the R. milleri C chromosome that did not appear to amplify or form a large puff. These differences can be related to developmental differences, in particular differences in cocoon construction between the two species.

Expression of calpastatin and myostatin genes associated with lamb meat quality.

Calpastatin (CAST) is an endogenous calpain inhibitor and its main function is to modulate the proteolytic action of enzymes responsible for post-mortem myofibril deterioration. The myostatin gene (GDF-8) acts as a negative regulator of skeletal muscle growth. The expression of these two genes, as well as their interaction, affects the quality of the meat, especially the tenderness phenotype. We evaluated the genetic groups Santa Inês, ½ Dorper-Santa Inês and ½ White Dorper-Santa Inês, slaughtered with 2.0 mm, 2.5 mm and 3.0 mm of fat thickness, comparing the levels of expression of the CAST and GDF-8 genes with the weight performance and carcass traits, especially the shear force values. We found significantly higher expression of myostatin and calpastatin in the Santa Inês genetic group. The ½ Dorper-Santa Inês genetic group had the lowest expression of these genes when slaughtered with 2.0 and 2.5 mm of fat thickness. In conclusion, the Santa Inês breed had the lowest phenotype values for meat tenderness, and the ½ Dorper-Santa Inês breed had the best performance for this characteristic. We suggest that high levels of the expression of the CAST and GDF-8 genes are associated with lower values of lamb meat tenderness, and that tenderness is related to the stage of muscular growth and development.

Relationship between cell proliferation and eruption rate in the rat incisor.

The aim of this study was to further define the relationship between cell proliferation and the rate of tooth eruption in the rat incisor. Vinblastine is a drug that blocks cellular mitosis and was used to inhibit cell proliferation in the odontogenic region of rat incisors that were submitted to a shortening treatment or to higher masticatory forces. Male Wistar rats were divided into five groups: normofunctional (control group for incisor eruption), hypofunctional (incisor submitted to eruption acceleration), hyperfunctional (incisors under higher masticatory forces), hypofunctional with vinblastine and hyperfunctional with vinblastine. In incisors submitted to shortening procedures, a significant decrease in the eruption rate and cell proliferation was observed two days after vinblastine injection, suggesting that incisor eruption is dependent on cell proliferation.

Development of Loxosceles intermedia Mello-Leitão (1934) (Araneae, Sicariidae) genital tract.

We examined the post-embryonic development of the male and female genital apparatus of the brown spider, Loxosceles intermedia. The development of the genital apparatus for both sexes begins with the appearance of inner structures. In the male genital apparatus, formation of the testes occurs first, followed by differentiation of the duct, ampulla and vas deferens, and finally the formation of the genital opening and differentiation of the copulatory organ (secondary sexual characteristic). Similarly, the development of the female genital apparatus begins with the formation of the ovaries, followed by the appearance of oocytes in vitellogenesis, then the development of oviducts and uterus internus and, finally, the spermatheca. These data may be very important in further comparative studies on the development of the reproductive system of spiders.

Diversidade genética entre três linhagens de codorna selecionadas para produção de ovos / Genetic diversity among three lineages of quail, selected for egg production

A diversidade genética entre três linhagens de codorna (Coturnix japônica) foi avaliada utilizando-se a técnica de random amplified polymorphic DNA (RAPD). As linhagens selecionadas para produção de ovos foram identificadas como amarela, azul e vermelha por meio de anilhas no pé esquerdo. Seis primers de RAPD amplificaram 55 loci, os quais geraram padrão de bandas intensa e reproduzível em gel de agarose. Os resultados indicaram polimorfismos dentro e entre as linhagens. A similaridade de Jaccard média e o índice de diversidade Shannon revelaram alta diversidade dentro das linhagens de codornas. O teste de Mantel por meio do algoritmo unweighted pair-group method using arithmetic average (UPGMA) e a dispersão de coordenadas principais indicaram diferenciação genética significativa, embora em baixo nível. Os resultados sugerem que a diversidade genética dentro e entre as linhagens de codornas da Universidade Estadual de Maringá são promissoras para uso em programas de melhoramento.

The genetic diversity among three lineages of quail (Coturnix japonica) was evaluated by the random amplified polymorphic DNA (RAPD) technique. The lineages were selected for egg production and identified with a yellow, blue, or red ring fasten on their left foot. Six selected RAPD primers amplified 55 loci, which generated intense and reproducible bands on agarose gel. The results indicated polymorphism within and among the lineages. The Jaccard similarity average and the Shannon diversity index revealed high diversity values within the quail lineages. The Mantel test, unweighted pair-group method using arithmetic average (UPGMA) algorithm and dispersion of principal coordinates indicated significant genetic differentiation, although at low levels. Overall, the results suggest that the genetic diversity within and among the quail lineags from the State Universidade Estadual de Maringá are promising for use in breeding programs.

Efeito dos genótipos para alphaS1-caseína sobre as frações proteicas e lipídicas do leite de cabra / Effect of genotypes for alphaS1-casein on proteic and lipidic fractions in goat milk

O alto polimorfismo encontrado no lócus do gene da αS1-caseína em caprinos, classificado em quatro níveis de expressão - alto, médio, baixo e nulo -, está associado à produção de 3,6; 1,6; 0,6 e 0g/L/alelo, respectivamente. O estudo foi realizado para investigar possíveis variações na produção de leite e seus constituintes, no perfil de caseínas e na lipólise da gordura. Quarenta e quatro cabras foram distribuídas em cinco genótipos: dois homozigotos, um para alta (AA) e outro para produção intermediária (EE), e três heterozigotos chamados AE, AF e EF, para αs1-caseína. Para a lipólise, o leite foi subamostrado em quatro alíquotas que sofreram tratamento térmico no momento da ordenha e após 24h de resfriamento. Diferenças entre genótipos foram observadas para a produção de caseína e de suas frações. As demais variáveis não diferiram entre genótipos. O genótipo AA apresentou os maiores conteúdos de caseína (28,6g/L) e de αS1-cn (22,3 por cento). Os demais genótipos apresentaram média de 20,4g/L. Os grupos AE e AF apresentaram média de 12,1, EE-10,1 e EF-9,1 por cento de αS1-cn. O resfriamento do leite por 24 horas aumentou a taxa de lipólise no leite. A genotipagem das cabras para αS1-cn pode ser usada como ferramenta de seleção com objetivo de obter produtos lácteos com distintos perfis de proteínas.

A high polymorphism is found in the locus of goat αS1-casein gene and it is classified in four levels of expression, named high, medium, and low, associated with production of 3.6, 1.6, 0.6, and 0 g/L/allele, respectively. The study was conducted to investigate possible variations on milk yield and components, profile of casein, and lipolysis of fat. Forty-four goats were assigned to five distinct genotypes as two homozygous, one for high (AA) and the other for intermediate yield (EE); and three heterozygous named AE, AF, and EF for the αs1-casein. For lipolysis, milk was sampled in four aliquots which were treated soon after milking and 24 hours after cooling. Differences were observed for both casein yield and its fractions. No difference was found for other variables. The AA genotype presented the higher content of both casein (28.6g/L) and αS1-cn (22.3 percent). Other genotypes averaged 20.4g/L for casein content. Values of αS1-cn were 12.1 percent for heterozygous and 10.1 and 9.1 percent for both EE and EF genotype respectively. Cooling the milk for 24 hours increased the rate of lipolysis. Genotyping goats for the αS1-cn can be used as a tool for selecting animal targeting milk products with distinct profiles of proteins.

Genetic evaluation of the HSP70 protein in the Japanese quail (Coturnix japonica).

Heat stress is one of the main problems in modern aviculture, since it affects birds especially in the final phase of rearing, causing bird mortality and economic losses to the aviculturist. The quail, as most birds, has difficulties in dissipating heat. However, little is known about the mechanism that controls the responses of the organism to stressor agents. Therefore, the study of heat shock proteins (HSPs) in these birds is important. A 960-bp portion of HSP70 was amplified using oligonucleotide primers specific for chickens. The fragment was sequenced, since it was the same protein, although some modifications have been observed. It showed 98% homology with HSP70 stress protein in Gallus gallus and 99% homology with Numida meleageris.

Genetic evaluation of the HSP70 protein in the japanese quail (Coturnix japonica)

Heat stress is one of the main problems in modern aviculture, since it affects birds especially in the final phase of rearing, causing bird mortality and economic losses to the aviculturist. The quail, as most birds, has difficulties in dissipating heat. However, little is known about the mechanism that controls the responses of the organism to stressor agents. Therefore, the study of heat shock proteins (HSPs) in these birds is important. A 960-bp portion of HSP70 was amplified using oligonucleotide primers specific for chickens. The fragment was sequenced, since it was the same protein, although some modifications have been observed. It showed 98% homology with HSP70 stress protein in Gallus gallus and 99% homology with Numida meleageris.

Novos polimorfismos no gene da obesidade em raças divergentes de suínos / Polymorphisms in the leptin gene in divergent swine breeds

Investigou-se a existência de polimorfismo no gene da leptina (gene da obesidade) entre varrões da raça nativa Piau (porco tipo banha) e matrizes mestiças de raças comerciais (Landrace/Large White e Landrace/Large White com Pietrain), selecionadas para peso e precocidade. Oito pares de primers foram desenhados a partir da seqüência disponível no GenBank (U66254), usada, neste trabalho, como seqüência de referência. Amostras de DNA foram extraídas de células sangüíneas brancas utilizando-se solução de fenol:clorofórmio, após tratamento com proteinase K. Os fragmentos gerados por amplificação da reação em cadeia da polimerase foram purificados e seqüenciados em seqüenciador automático. As seqüências de nucleotídeos, obtidas a partir do DNA das raças comerciais de suíno, apresentaram maior similaridade com a seqüência de referência, e as seqüências geradas a partir do DNA dos animais nativos divergiram de ambas em algumas posições. Dos 28 polimorfismos encontrados, oito foram observados em apenas uma das três seqüências geradas a partir do DNA das raças nativas. Doze estavam presentes em duas seqüências, e os oito polimorfismos restantes foram encontrados nos três animais nativos.

Leptin gene (obese gene) polymorphism was investigated in Piau boars (a fat, native breed) and sows from commercial strains (Landrace/Large White and Landrace/Large White by Pietrain) chosen for rapid growth and early sexual maturity. Eight pairs of primers designed using the sequence available from GenBank (access n° U66254) were identified as the reference sequence in this project. DNA samples were extracted from white blood cells using phenol:chloroform solution, after treatment with proteinase K. Fragments generated by amplification of the Polymerase Chain Reaction were purified and sequenced in an automatic sequencer. Nucleotide sequences obtained from DNA of commercial swine breeds were similar to the reference sequence; whereas sequences generated from native breed DNA diverged from the reference sequence and from domestic breed DNA. Of the 28 polymorphisms found, eight were observed in only one of the three sequences generated from DNA of native breeds. Twelve polymorphisms were present in two sequences and the eight remaining polymorphisms were found in all three categories of DNA.

Circadian variation of the cell proliferation in the jejunal epithelium of rats at weaning phase.

Circadian variation in cell proliferation of the jejunal epithelium of 18-day-old rats was studied using the 2-h arrested metaphase score and crypt isolation method. A continuous decrease in the arrested metaphases occurred from 07.00 h to 13.00 h. From 17.00 h arrested metaphase values increased and were maintained at the higher level during the dark period as showed by Cosinor analyses (P < 0.05). These results indicate that in the young rat there is already a circadian variation in jejunal epithelial cell proliferation as early as 18 days. We can even suggest that the presence of a circadian rhythm at weaning contributes to the steady state of cell proliferation in the intestinal epithelium observed in adult life.

Cell cycle time and rate of entry of cells into mitosis in the small intestine of young rats.

Cell cycle time (T(C)) and the rate of entry of cells into mitosis (r(M)) in the jejunum and duodenum of young rats were investigated using the stathmokinetic method. The cell cycle times in the jejunum were 24.3 and 28.3 h in light and dark periods, respectively. Cell cycle times in the duodenum were 17.1 and 21.5 h in light and dark periods, respectively. Rates of entry of cells into mitosis in the jejunum were 1.2 and 1.1 cells/cell/h in light and dark periods and rates of entry of cells into mitosis in the duodenum were 1.4 and 1.8 cells/cell/h in light and dark periods, respectively. Although these changes to cell cycle time values are not statistically significant, the variation between the two periods should be considered in relation to its possible biological effects.

Analysis of the amplification and transcription of the C3-22 gene of Rhynchosciara americana (Diptera: Sciaridae) in transgenic lines of Drosophila melanogaster.

Drosophila melanogaster was transformed with an 18 kb fragment of the C3 DNA puff of Rhynchosciara americana, including the C3-22 gene and the origins of replication that direct amplification. Different tissues and developmental stages of five independent transgenic lines were analyzed by quantitative Southern blot hybridization. No indication was found that the transformed fragment was amplified, strongly suggesting that factors involved in DNA puff amplification have not been conserved in Drosophila. Transcription of the C3-22 gene in the transgenic lines was found to be at a low and constitutive level throughout development. These results indicate that, unlike other DNA puff genes, the factors that regulate the C3-22 gene are not conserved in Drosophila.